The investigation of the fine structure of living organisms has heretofore been limited by the optical transparency of the biological specimens. Whole mounts of transparent, unstained organisms can be examined under dark field illumination as well as ordinary bright field illumination with phase contrast or differential interference contrast. These methods can be used in conjunction with vital dyes and can also give gross dynamical information concerning muscular contractions, cell movements and shape changes which occur in the course of development of the organism. For detailed structural information, especially of embryonic development in optically opaque organisms, routine histological methods of sectioning and staining are generally used, and dynamic changes have to be inferred by making comparisons between sections prepared at successive stages in the development of the organism.
Polarized light microscopy is a technique which has traditionally been used for the investigation of crystalline structures of minerals and various "inert" biological materials, such as fibres, bone, chitin and exoskeletons, and, in a few cases, fixed sections of organisms. The technique has also been used in the study of contractions in isolated muscle fibres, before being supplanted by low-angle X-ray diffraction. In general, conventional polarized microscopy gives static ultrastructural information concerning the arrangement of atoms in the molecules, i.e. their birefringence or anisotropy, as well as the ordered arrangement of molecules in an array.
In order to appreciate the methods used in accordance with the present invention it is necessary to understand how a polarizing microscope functions. The essential parts of a polarizing microscope are shown schematically in FIG. 1. Conventionally, a sample (object) 10 is observed using a light source 12 and a single polarizer 14 or a polarizer 14 and analyzer 16 (i.e. a second polarizer) whose vibrational directions are crossed at right angles (90.degree.). Interference colours are obtained from highly birefringent objects when white light is used as the light source 12 and with the polarizers 14, 16 crossed at right angles.
Each polarized (monochromatic) light ray, on passing through the birefringent object 10, is split into two mutually perpendicularly vibrating slow and fast rays, as shown in FIG. 2, which propagate through the object 10 at different velocities.
The retardation of the slow ray relative to that of the fast ray, hereinafter referred to as relative retardation, measured in nanometers (nm), generates a phase difference between the rays as they emerge from the object 10. In FIG. 2, the following convention is used to represent the vibrational directions of the light ray: a ray vibrating in the plane of the Figure is drawn as a line crossed by short bars at 90.degree., one vibrating perpendicularly to the plane of the Figure is drawn as a line with dots, one vibrating at 45.degree. to the plane of the Figure is drawn as a line crossed by short bars at 45.degree., and one vibrating at 135.degree. to the plane of the Figure is drawn as a line crossed by short bars at 135.degree.. The state of polarization of the light ray from the light source 12 is random. After passing through the polarizer 14, the ray 23 is polarized with its vibrational direction at 45.degree. to the vibrational directions of the birefringent object. 10. Within the birefringent object 10, the ray is split into two rays 24 and 25 vibrating perpendicularly to each other. On emerging from the object 10, a phase difference has been introduced between the slow ray 25, continuing as ray 27, and the fast ray 24, continuing as ray 26. When slow and fast rays are recombined into a single ray 28 by passage through the analyzer, they interfere either destructively (as shown in FIG. 2) or constructively, depending on the phase difference introduced by the birefringent object 10. The phase difference will also differ according to the wavelength of the light involved. Thus, when white light consisting of a full spectrum of wavelengths in the visible range--from 390 nm to 780 nm--is used, the phase difference introduced will differ in different parts of the spectrum. Different interference colors (or polarization colors) will be obtained according to the relative retardation between the slow and fast waves introduced by the birefringence of the object. Colored light is simply white light missing certain parts of its complete spectrum.
In the known crystallographic systems, in order to determine the vibrational directions of the slow and fast waves, and hence the anisotropy of the crystalline structure of the object 10, compensating birefringent crystal plates are inserted between the object and the analyzer. Such a compensator crystal plate is indicated at 18 in FIG. 1. The compensator plate is placed with its slow (and fast) vibrational directions at 45.degree. between those of the crossed polarizers to align with those of the object 10 so that the birefringence of the object can be estimated from the color changes observed in the specimen 10 according as to whether the relative retardations are added, in the case where the vibrational directions of the fast and slow waves in the sample and the compensator are aligned with each other, subtracted, in the case where the fast and slow wave directions in the sample and the compensator are 90.degree. out of alignment.
When the birefringence of the sample is not sufficiently great to generate interference colors by itself, i.e. when the relative retardation introduced between the fast and slow polarized light rays is less than 350 nm, interference colors are conventionally generated by the addition of a compensator plate with its vibrational directions aligned at 45.degree. to those of the crossed polarizers. However, for weakly birefringent materials, i.e. those in which the relative retardation introduced is less than 100 nm, there has not heretofore been a method for establishing interference colors.
It has recently been established that a high degree of dynamic order exists in living organisms, in that coherent regimes, associated with non-equilibrium phase transitions, can arise in macromolecules and macroscopic arrays of molecules under certain energetic conditions. It should therefore be possible to observe such regimes of dynamic order within living organisms. It is already known, for example, that liquid crystals can assume ordered states at certain temperatures and under the influence of electric fields. Many arrays of macromolecular structures, as for example biological membranes and muscle fibres, may have properties not unlike those of liquid crystals.